Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation

• Published in: Journal of cell science Vol. 114; no. Pt 13; pp. 2437 - 2447
• Main Authors: Brock, R, Jovin, T M
• Format: Journal Article
• Language: English
• Published: England 01.07.2001
• Subjects: Animals; CHO Cells; Cricetinae; Culture Techniques; ErbB Receptors - metabolism; Green Fluorescent Proteins; Image Processing, Computer-Assisted; Intramolecular Transferases - metabolism; Kinetics; Luminescent Proteins - metabolism; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Signal Transduction; Subcellular Fractions; Time Factors; Tumor Cells, Cultured
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.114.13.2437

Epidermal growth factor receptor (EGFR, erbB1) activation and translocation of the Shc adaptor protein to activated receptors were analyzed at the subcellular level by dual-label immunofluorescence and confocal laser scanning microscopy in conjunction with a new microsphere-based protocol. In the Quantitative Microsphere Recruitment Assay (QMRA) introduced here, epidermal growth factor-coated 1 microm diameter microspheres were distributed over the surface of adherent tissue culture cells expressing the receptor. High-resolution confocal microscopy of a fusion construct of the receptor and the green fluorescent protein expressed in Chinese hamster ovary cells demonstrated that engulfment and internalization of the microspheres occurred rapidly within minutes, and in a receptor activation-dependent manner. In human epidermoid carcinoma A431 cells, receptor activation and Shc translocation persisted over the 20-minute time course of the experiments. However, at the subcellular level the positive correlation of receptor activation and Shc translocation observed at 5-8 minutes dissipated, indicating a time-dependent decoupling of the two events and variation in the kinetics of signal transduction for different subcellular locations.